CONFORMATIONAL STUDIES OF PROTEINS IN CONTACT ACTIVATION

该项目的长期目标是了解 负责表达四种生物活性中的每一种 参与凝血接触相的蛋白质。这个 它们相互作用的构象和结构方面将是 使用各种生物物理方法进行研究，如圆二向色性， 溶剂微扰、紫外差光谱、光散射、 沉淀、化学修饰、分子模型构建和 蛋白质二级结构的预测。 具体地说，目标是：(1)因子XII中的多肽序列 已知参与表面结合的化合物将被合成。 这些多肽对应于因子XIII-28和因子XII134-153 完整的XII因子将通过氨基酸取代和 特定的化学修饰。系统动力学和构象 将进行分析，以确定各种形式的修改 会影响抑制因子XII的激活，通过 表面带负电。(2)含有GHKHER的多肽和 Hmwk中已知参与表面结合的HGLGHGH序列 将被合成。HMWK中的这些已知涉及 表面结合将被合成。这些独特的序列，它们 对应于完整HMWK的HMWK426-431、HMWK441-447和HMWK451-457 将通过氨基酸取代和特定的化学物质进行修饰 修改。系统的动力学和构象分析将 研究电荷和氨基酸的各种变化 取代作用会影响多肽抑制hmwk的能力。 与PK和因子XI的结合将被合成。这些多肽，它们 对应于HMWK556-595和HMWK556-613的完整HMWK将 通过氨基酸取代和化学修饰进行修饰。系统化 将进行动力学和构象分析，以调查 多肽的各种修饰方式将影响其作用能力。 抑制hmwk与这两种酶原的结合。(4)最后，使用 化学交联剂与合成肽结合， HMWK556-595和HMWK556-613，以便建立和定义HMWK PK和凝血因子XI的结合部位。

The long term goal of this project is to understand the mechanism that are responsible for the expression of biological activities of each of the four proteins involved in the contact phase of blood coagulation. The conformational nad structural aspects of their interactions will be investigated using various biophysical methods, such as circular dichroism, solvent perturbation, uv difference spectroscopy, light scattering, sedimentation, chemical modification, molecular model building and prediction of protein secondary structure. Specifically, the objectives will be: (1) Peptide sequences in factor XII which are known to be involved in surface binding will be synthesized. These peptides, which correspond to factor Xiii-28 and factor XII134-153 of the intact factor XII will be modified by amino acid substitution and specific chemical modifications. Systematic kinetic and conformational analysis will be undertaken to determine how various forms of modifications of the peptides will affect the inhibition of factor XII activation by negatively charged surfaces. (2) Peptides containing the GHKHER and HGLGHGH sequences in HMWK which are known to be involved in surface binding will be synthesized. These in HMWK which are known to be involved in surface binding will be synthesized. These unique sequences, which correspond to HMWK426-431, HMWK441-447 and HMWK451-457 of the intact HMWK will be modified by amino acid substitution and specific chemical modifications. Systematic kinetic and conformational analysis will be conducted to investigate how various alterations in charges and amino acid substitutions will affect the ability of the peptides to inhibit HMWK binding to PK and factor XI will be synthesized. These peptides, which correspond to HMWK556-595 and HMWK556-613 in the intact HMWK will be modified by amino acid substitution and chemical modifications. Systematic kinetic and conformational analysis will be undertaken to investigate how various ways of modifications of the peptides will affect the ability to inhibit the binding of HMWK to the two zymogen. (4) Finally, to use chemical crosslinking reagent, in conjunction with the synthetic peptides, HMWK556-595 and HMWK556-613, in order to establish and define the HMWK binding site in PK and factor XI.